1. Field of the Invention
The present invention relates to a method for preparing an antibody, particularly to a method for preparing an antigen-specific antibody by constructing a library of phage-displayed single chain variable fragment (ScFv) of an antibody using a novel frame-shifting PCR step.
2. Description of the Related Art
Monoclonal antibody (mAb) is mainly derived by cell fusion as described originally by Kohler and Milstein (1975). Owing to the broad application and wide perspective of the monoclonal antibody, important advances in design, selection, and production of engineered antibodies have been made. The traditional method for recombinant mAb construction like hybridoma technology has many limitations, such as duration, stability and class manipulation (Harlow and Lane, (1988) Monoclonal antibody: A laboratory manual pp. 141–149). Although new technology like the display of antibody fragments on the surface of filamentous phages and the subsequent selection of antibodies have been proved as an effective tool for the isolation of antigen specific antibodies (Barbas, C. F., Kang, A. S., Lerner, R. A. & Benkovic, S. J. (1991), Assembly of combinatorial antibody libraries on phage surfaces: the gene III site, Proc. Natl. Acad. Sci., USA 88, 7978–7982; Marks, J. D., (1992), By-passing immunization: building high affinity human antibodies by chain shuffling, Bio/Technology, 10, 779–783; Nissim, A. (1994), Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents, EMBO J. 13, 692–698), it still has limitations. The library of phage-displayed single chain variable fragment (ScFv) of the antibody has been used for deriving tailor-made antigen-specific monoclonal antibody in the last decade. Furthermore, affinity enhancement of ScFv can be achieved by in vitro mutation.
In general, the affinity of isolated antibodies is proportional to the initial size of the library used for selection. Using mRNA as an enriched source of expressed and spliced antibody genes neglects the allelic exclusive genes so as half of the potential genes. Hence, the diversity of the library used for selection becomes limited. Moreover, the gene loss will happen in both self-intolerance gene elimination and gene inactivation during maturation of B-lymphocytes. Finally, the other problem that needs to be considered is the existence of non-functional genes. These genes refer to Ab genes containing stop codon(s) in their segments, either naturally or created by the vague rearrangement processes during the somatic recombination.